Lipoprotein lipase (LPL) is required for uptake of triacylglycerol by tissues from plasma lipoproteins. It is synthesized in parenchymal cells and transferred to capillaries where it acts. Active LPL is a noncovalent homodimer of N-linked glycosylated subunits and has a high affinity for heparin. The relation between glycosylation, dimerization, affinity for heparin, and activity of LPL was studied in cultured brown adipocytes of newborn mice. It was found that normal cells contained several forms of glycosylated LPL. The most prevalent form was dimeric with high heparin affinity and catalytic activity. Less prevalent forms were monomeric, dimeric, and higher oligomeric, all with low heparin affinity and no activity. Findings in cells treated with 1-deoxymannojirimycin demonstrated that dimerization, high heparin affinity and activity of LPL did not requite processing of oligosaccharides in Golgi. Cells treated with tunicamycin synthesized unglycosylated LPL which was dimeric, had low heparin affinity, and was inactive. Blockade of glucosidase I in endoplasmic reticulum (ER) of normal cells with castanospermine (CST) had no effect on dimerization or acquisition of high heparin affinity. However, it prevented processing of LPL to an active form. Hence, one- fourth of LPL in CST-treated cells was dimeric and had high heparin affinity, but was inactive. Earlier studies showed that CST caused retention of LPL in ER. Adipocytes cultured from cld/cld mice synthesized fully glycosylated LPL which was dimerized, had high heparin affinity, and was trimmed of sugar residues in ER, but the lipase was inactive and retained in ER. Translocation of Golgi components to ER by brefeldin A resulted in synthesis of active LPL and processing of one oligosaccharide per subunit to endo H-resistance in CST-treated and cld/cld cells. The findings indicate that LPL synthesized in cld/cld cells is capable of being processed to active lipase and the enzymes needed for activation of LPL are present in Golgi, but the cells are unable to transport LPL to Golgi. We conclude that high heparin affinity of LPL requires glycosylation and dimerization but not trimming of oligoglycans in ER, whereas catalytic activity of the lipase requires high heparin affinity, trimming in ER, and some modification other than oligoglycan by Golgi, in addition to glycosylation and dimerization. Furthermore, glycosylation, dimerization and acquisition of high heparin affinity occur in ER, while activation occurs in Golgi. Schematic models were developed for the four LPL dimers found in cells.